Transcription in bacteria is carried out by a multi-subunit RNA polymerase (RNAP) that is conserved from bacteria to humans. Promoter-specific transcription initiation depends on the ??subunit of RNAP, which must associate with the catalytically proficient core enzyme to form the holoenzyme. Historically defined as an initiation factor, ? can also remain associated with the transcription elongation complex and influence the transcription process during elongation. In particular, ? can induce early elongation pausing by establishing sequence-specific interactions with promoter-like DNA sequence elements within the initial transcribed region of a gene. The proposed research builds on the demonstration that such promoter-like pause- inducing elements can function to inhibit ? loss during the earliest stage of elongation, increasing the ? content of elongation complexes throughout the transcription unit. The results of bioinformatic analyses suggest that promoter-proximal ?-dependent pause elements are associated with at least 20% of all E. coli promoters, which implies that this mechanism for regulating the ? content of elongation complexes is likely a general feature of transcription in E. coli. The first aim of the proposed research is to investigae the effects of ? retention on E. coli gene expression. The second aim is to investigate whether the potential roles for ? during transcription elongation extend beyond the primary ? factors and to determine whether different types of ? factors can participate in initiation and elongation on the same transcription unit (? cross-regulation). The third aim is to investigate the mechanism by which promoter-proximal pause elements influence the ? content of downstream elongation complexes. The fourth aim is to investigate whether a specific ?-core interaction that may be essential for cell viability facilitates the release of ? from the DNA in the context of a paused early elongation complex. Together, the proposed experiments will lead to a deeper understanding of the roles of a classical initiation factor during transcription elongation, contributing to an accumulating body of evidence for the importance of post-initiation regulatory events in both prokaryotes and eukaryotes.